Precautions for Use of C18 Liquid Chromatography Column

Using a C18 liquid chromatography column effectively requires attention to several precautions to ensure accurate and reliable results. Here are some key precautions for the use of C18 liquid chromatography columns:

  1. Column Conditioning:

    Before the initial use or after storage, it’s essential to condition the C18 column with the mobile phase to stabilize its performance and remove impurities.

  2. Mobile Phase Compatibility:

    Ensure that the mobile phase is compatible with the C18 stationary phase. For reversed-phase chromatography, the mobile phase typically includes water and an organic solvent (e.g., acetonitrile or methanol).

  3. pH Compatibility:

    Be mindful of the pH of the mobile phase. C18 columns are generally stable within a specific pH range. Extreme pH values can lead to degradation of the stationary phase.

  4. Avoid Contaminants:

    Keep the mobile phase and sample solutions free from contaminants that could interfere with the analysis. Use high-quality solvents and filters to prevent particulate matter.

  5. Sample Solubility:

    Ensure that the samples are fully soluble in the mobile phase to avoid precipitation or sample carryover issues. If necessary, consider adjusting the sample solvent composition.

  6. Guard Columns:

    Consider using guard columns to protect the analytical C18 column from particulate matter and strongly retained compounds. Guard columns are replaceable and help extend the life of the analytical column.

  7. Column Temperature:

    Maintain a consistent column temperature if possible. Temperature fluctuations can affect chromatographic performance. Some separations benefit from temperature control for improved resolution.

  8. Flow Rate and Pressure:

    Operate the column within the recommended flow rate and pressure limits. Exceeding these limits can lead to column damage, loss of efficiency, or compromised separation.

  9. Avoid Extreme Conditions:

    Avoid exposing the C18 column to extreme conditions, including high temperatures, harsh solvents, or conditions outside the specified pH range.

  10. Regular Maintenance:

    Perform regular maintenance, including flushing the column with a compatible mobile phase and replacing the guard column if used. Follow the manufacturer’s recommendations for column care.

  11. Column Storage:

    When not in use, store the C18 column in a recommended mobile phase to prevent drying and preserve the integrity of the stationary phase.

  12. Column Selection:

    Choose the appropriate C18 column dimensions, particle size, and pore size for the specific analytical requirements. Consider factors such as sample complexity, analyte properties, and desired separation efficiency.

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  1. Read the manual before using:

    What solvents are stored inside the C18 chromatographic column (C18 Low PH HPLC Column, C18 Alkaline HPLC Column, C18-Aqueous HPLC Column, ODS C18 HPLC Column) must be read clearly, so the manual of the chromatographic column must be read clearly, of course, it also includes the maintenance regulations recommended in the manual, which are very useful to us? For reversed-phase columns, the solvent is basically acetonitrile or methanol. But the normal phase column is different because, in addition to the normal phase system, the normal phase column can also be applied to the reverse phase system. Therefore, you must see clearly whether you need to replace the solvent inside the column for your method.

  2. Rinse the column with pure water to flush out the buffer:

    Using a strong solvent to flush the chromatographic column will cause great damage to the internal packing, especially pure water unless your chromatographic column is water-resistant. The method of flushing the chromatographic column is to flush with the water system and organic system of the mobile phase ratio. If you think that the mobile phase ratio cannot clean the chromatographic column or the organic system occupies too much of this ratio, you can use pure water containing about 10% to rinse.

  3. The new column stored in acetonitrile or methanol can be used directly:

    Theoretically, the liquid chromatography column is different from the gas chromatography column and needs to be activated, but because the organic system stored in the liquid chromatography column is volatile, it should be flushed with the same solvent with a small flow rate, and the inside of the column for a certain period of time before use.

  4. The column should be stored in pure acetonitrile or methanol:

    If the storage liquid inside the chromatographic column is volatile, so in order to reduce its volatilization rate, you can add 5%-10% water, which can reduce the volatilization of some organic systems to a certain extent. If we add another insurance, we can seal it with parafilm after blocking the plug. In the same way, after the chromatographic column is not used for a period of time, the chromatographic column should be flushed with a small flow rate.

  5. After the C18 liquid chromatography column is contaminated, remove the sieve for ultrasonic cleaning:

    For the pollution of the chromatographic column, the sieve plate is the first to bear the brunt. Therefore, our common method is to remove the column head and take the sieve plate to ultrasonic cleaning.6. Since the stationary phase of the C18 column (except for the polar modification treatment) has strong hydrophobicity, try not to use high water phase conditions during use. If the water ratio is too high, it will easily cause the stationary phase to collapse and cause the column efficiency to decrease and irreversible negative effects, due to the high carbon loading. It is recommended that the water ratio during use should not exceed 90%.7. The pressure of the chromatographic column increases and the column performance decreases
    Usually, the chromatographic column is contaminated, and regenerating the chromatographic column can restore part of the column’s efficiency.

  6. Sudden increase in column pressure:

    Usually, it is caused by salt precipitation or clogging of the sieve plate. If salt is precipitated, flush with 10% methanol-water at a low flow rate until the pressure is restored; if it is judged that it is not salt precipitation, use pure methanol or 10% methanol-water as the flow instead of flushing the chromatographic column (reversely connect the chromatographic column without connecting the detector). If the backflush has no effect for half an hour, it means that the backflush is invalid, and other reasons should be found.